Rationale: The effective treatment of life-threatening fungal infections in humans is a major unmet clinical challenge. Annotated genomic sequences of the major human fungal pathogens are now available, creating an opportunity to revolutionize medical mycology through the application of systematic approaches. In particular, as genome-wide knockout collections in model yeasts have been instrumental to the dissection of fundamental eukaryotic cellular processes, the generation and analysis of analogous gene deletion collections in pathogenic fungi is now beginning to allow the systematic elucidation of the molecular determinants of virulence in the mammalian host. Cryptococcus neoformans is one of the three most important human fungal pathogens in humans. It is highly tractable experimentally, having a complete sexual cycle amenable to genetics and outstanding animal models for infection. This opportunistic encapsulated budding yeast is the most common cause of fungal meningitis. Annually, there are estimated 1,000,000 cases that result in ~600,000 deaths, and one-third of deaths in AIDS patients are attributed to this one pathogen. Using optimized methods for gene targeting, our laboratory constructed a library 1201 gene deletion strains. We exploited this resource for systematic screens of pathogen fitness in experimental mice, expression of known virulence factors, mechanisms of hypoxic adaptation, and mechanisms of phagocytosis-inhibition. However, many of the genes identified in these screens, while critical for pathogenicity, are of unknown molecular function. Thus, obtaining further insigh into virulence requires methods that can lead to the functional annotation of novel genes. Objective: We propose to address the problem of functional annotation by applying chemogenetic profiling, a powerful chemical-genetic method. In this approach, the impact of chemical perturbations on the fitness of large numbers of defined gene knockouts is quantified. The resulting phenotypic fingerprints are used to cluster genes into functional groups and to define their roles with respect to each other. We propose to apply this method to C. neoformans to functionally annotate key genes necessary for pathogenicity. To accomplish this goal, we will first develop a project informatics foundation and to identify bioactive chemicals suitable for chemogenetic profiling. We will then obtain and analyze full-dilution quantitative fitness responses to an array of bioactive chemicals. Finally, we will exploit the results to develop and test concrete hypotheses for the role of gene products of unknown function that we have previously implicated in pathogen fitness in the host. Impact: The proposed work will produce the first detailed phenotypic map of a large portion of the genome of any human microbial pathogen. By clustering genes with similar profiles together, these studies are anticipated to lead to critically-needed insight into the molecular functions of pathogen factors identified in ou previous studies of mammalian infection. In addition, this work is anticipated to define chemical modulators of essential virulence pathways, which are expected to be powerful tools for studies of pathogen biology and future therapeutic development. PUBLIC HEALTH RELEVANCE: The diagnosis and treatment of serious fungal infections of humans is a major unmet clinical challenge. Cryptococcus neoformans is among the most important fungal pathogens, accounting for approximately one-third of worldwide deaths from HIV/AIDS. By combining small molecules and genetics, this study will decipher the function of pathogen genes required for success in the host whose molecular functions are currently unknown. This knowledge is anticipated to produce a new foundation for the development of much-needed therapeutics.